Biology subjects

Bednarz, B.

Publications and source records attributed to Bednarz, B..

7 recordsLinked to original sources

bioRxiv2026

Optimized dosing of spatially heterogeneous radiopharmaceutical therapy reprograms poorly immunogenic tumors to enhance response to immune checkpoint blockade

Immune checkpoint inhibitors (ICIs) are ineffective in patients with poorly immunogenic tumors due to exclusion or exhaustion of effector T cells in the tumor microenvironment (TME) and/or inadequate priming of tumor-specific T cells. Preclinical and clinical studies suggest that radiopharmaceutical therapy (RPT) may overcome such challenges, but an optimal approach to RPT dosing has not been clarified in combination with ICIs. Conventional RPT dosimetry emphasizes mean absorbed radiation doses in tumors and normal tissues, neglecting the spatial distribution of this dose in the TME. We hypothesize that RPT dose heterogeneity in tumors may be leveraged to initiate spatially restricted immuno-radiobiological effects in TME that potentiate antitumor immunity when injecting an activity that delivers high dose (V10%>20 Gy) while also preserving low dose regions (V90%<2.5 Gy) in the TME and not inducing systemic lymphopenia. In studies using lutetium-177 RPT in combination with ICIs, administered activities that met these dosimetric optimization metrics occurred at different mean tumor doses for different RPT agents and different models, yet each achieved therapeutic efficacy in poorly immunogenic murine tumors. Spatial transcriptomics at day 7 after RPT revealed heterogeneous distribution of tumor cell markers with distinct dose-response profiles and localization of dendritic cell (DC) migratory markers in regions of moderate/high dose response marker expression. Combination therapy sustained conventional type 1 DC (cDC1) activation in tumor draining lymph nodes and promoted systemic expansion of antigen-specific CD8 T cells, while serial biopsies revealed effector T cell recruitment and inflammatory cytokine expression in the TME. Optimized intratumoral dose heterogeneity may enable and predict response to ICIs when combined with RPT in tumors not responding adequately to ICIs alone.

cancer biology↗

bioRxiv2026

Intratumoral dose heterogeneity promotes adaptive anti-tumor immunity and predicts clinical response to radiopharmaceutical therapy

Radiopharmaceutical therapies (RPT) deliver non-uniform radiation dose in tumors and the impact of this on response is poorly understood. Dose heterogeneity could engender treatment resistance in low dose regions, yet we hypothesize that a broader array of dose-dependent immuno-radiobiological mechanisms in tumor microenvironments (TME) and preservation of immune function in low-dose regions could promote adaptive anti-tumor immunity and response. In murine models, non-uniform lutetium-177 delivering <2.5 Gy to >20 Gy in a TME induced broader immunomodulatory effects and T cell-dependent survival improvement compared to more uniform distributions. Preserving low-dose regions promoted dendritic cell activation and TME infiltration of clonally expanded CD8+ T cells. In three independent cohorts of patients with prostate cancer, heterogeneous tumor dose distribution strongly correlated with improved clinical outcomes. These findings defy expected radiobiological dose-response and define a novel mechanism of action for RPT, supporting clinical investigation of dose distribution for optimizing patient selection and personalized dosing.

cancer biology↗

bioRxiv2026

Tuning the Structural Properties of a Single-Domain Antibody Scaffold for Improved Fibroblast Activation Protein Targeting

Fibroblast activation protein (FAP) is an attractive target for the development of cancer theranostics due to its selective expression on cancer-associated fibroblasts (CAFs). While a number of small-molecule FAP inhibitors (FAPIs) have been developed, few biologics have been investigated as FAP targeting vectors. Camelid-derived single-domain antibodies, or variable-heavy-heavy domains (VHHs), offer a compelling alternative, combining high affinity with versatile engineering options. In this study, we first identified a novel anti-FAP VHH, F7, from an affinity-matured camelid phage display library. To investigate how valency and molecular weight affected target engagement and in vivo properties, F7 was engineered into three formats: a monomer (F7), a tethered dimer (F7D), and an Fc-fusion protein (F7-Fc). All three were specific for FAP with the two bivalent constructs demonstrating picomolar affinity. Positron emission tomography imaging in FAP-positive xenograft models revealed distinct pharmacokinetic profiles across constructs with notable differences in tumor uptake and clearance. F7 had rapid uptake and clearance resulting in significantly higher tumor uptake than FAPI-46. Low molecular weight bivalent F7D demonstrated similar kinetics but was retained by the tumor resulting in a high tumor-to-blood ratio with secondary uptake limited to clearance organs. The largest construct, F7-Fc, resulted in the highest tumor uptake and allowed for longitudinal imaging. Absorbed dose calculations confirmed that tumors received significantly higher radiation doses compared to normal tissues. These findings demonstrate that tuning VHH scaffold size and valency can improve biodistribution and retention, establishing F7-based constructs as promising targeting vectors for FAP.

cancer biology↗

bioRxiv2026

A MET-Targeted Variable New Antigen Receptor Theranostic for Non-Small Cell Lung Cancer

The MET receptor tyrosine kinase is mutated or amplified in [~]6% of non-small cell lung cancer (NSCLC) and overexpressed in [~]80% of all NSCLC cases. A theranostic agent that can both see and treat MET-altered NSCLC has never been described before in the literature. Here, we report a shark-derived single-domain variable new antigen receptor (VNAR) for MET with theranostic applications. Following the immunization of a juvenile nurse shark (Ginglymostoma cirratum) with the extracellular domain of human MET, we identified a VNAR clone that specifically engaged MET with high affinity. Engineering the lead VNAR into a bivalent human Fc, vMET1-Fc, yielded a construct that selectively targeted and was internalized by MET-positive cells without affecting cell viability or downstream MET signaling. When radiolabeled with the positron emitting isotope Zr-89, [89Zr]Zr-vMET1-Fc enabled longitudinal PET/CT imaging. High tumor uptake with low background was observed in MET-positive NSCLC xenografts administered [89Zr]Zr-vMET1-Fc. As a targeted beta-particle radiotherapy, [{superscript 1}Lu]Lu-vMET1-Fc resulted in marked tumor-growth delay and exhibited a favorable toxicity profile, collectively improving progression-free survival in NSCLC mouse models. Non-human primate PET/CT imaging studies with ([Zr]Zr-vMET1-Fc in healthy rhesus macaques confirmed favorable biodistribution and dosimetry, predictable clearance, and minimal off-target uptake. Additional blood chemistry analysis found no significant immune response or cytotoxicity. Together, these findings establish vMET1-Fc as a theranostic agent for imaging and treating MET-altered NSCLC. Statement of SignificanceA shark-derived antibody selectively targeting MET shows preclinical efficacy as a theranostic agent for MET-altered cancer.

cancer biology↗

bioRxiv2024

Low Dose Radiation by Radiopharmaceutical Therapy Enhances GD2 TRAC-CAR T Cells Efficacy in Localized Neuroblastoma

BackgroundWhile chimeric antigen receptor (CAR) T cells have achieved significant success against hematological malignancies, efficacy against neuroblastoma has been limited. Virus-free CRISPR-edited GD2 TRAC-CAR T cells have been developed as a potential means of improving CAR T efficacy but are not curative. Radiopharmaceutical therapy (RPT) is a promising approach to enhance the effectiveness of immunotherapies, including immune checkpoint inhibitors. However, it remains unclear whether RPT can synergize with GD2 TRAC-CAR T cells to improve outcomes in neuroblastoma. MethodsDosimetry studies were conducted to measure the absorbed radiation dose delivered by lutetium-177 (177Lu) in both in vitro and in vivo models. Tumor-bearing mice were treated sequentially with low dose radiation by 177Lu-NM600, an alkylphosphocholine mimetic radiopharmaceutical agent, followed 9 days later by GD2 TRAC-CAR T cells generated in a virus-free manner by CRISPR/Cas9. Tumor burden was monitored through bioluminescence imaging and tumor size measurements. Mechanistic studies were performed using flow cytometry, multiplex assay and single-cell proteomic analysis. ResultsLow dose radiation delivered by 177Lu-NM600 synergized with GD2 TRAC-CAR T cells in a localized neuroblastoma model, resulting in complete tumor regression in all mice. The optimal combination was dependent on both the radiation dose and timing to minimize the negative impact of radiation on CAR T cell viability. Irradiation of neuroblastoma cells by low-dose RPT before GD2 TRAC-CAR T cells enhanced the release by CAR T cells of perforin, granzyme B and cytokines like TNF- and IL-7 while abrogating TGF-{beta}1 secretion. Additionally, low-dose RPT upregulated Fas on neuroblastoma cells, potentially enabling a CAR-independent killing. ConclusionsThis study demonstrates that low-dose RPT can enhance CAR T cell efficacy to treat a solid tumor. Findings suggest that optimization of radiation dose and timing may be needed for each patient and RPT to account for effects of varied tumor radiosensitivity and dosimetry. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/621668v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@153ff5dorg.highwire.dtl.DTLVardef@1a269b7org.highwire.dtl.DTLVardef@1ca9a53org.highwire.dtl.DTLVardef@59f461_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

The gamma-butyrolactone receptors ScbR and AtrA form a quorum sensing switch between coelimycin and actinorhodin synthesis in Streptomyces coelicolor A3(2)

BackgroundQuorum sensing enables gene expression regulation in response to changes in cell population density and controls diverse processes, such as biofilm formation, virulence and antibiotic production, in bacteria. In one of the largest, soil-dominant phylum Actinobacteria, cell-to-cell communication occurs through the small, membrane-diffusible signalling molecules gamma-butyrolactones (GBLs). Their actions are exerted through receptor proteins that also act as response regulators in a one-component system manner. With only a few GBL systems characterized, most of them come from the large, antibiotic-producer genus Streptomyces. In the model organism Streptomyces coelicolor A3(2), two GBL receptors, ScbR and SlbR, which are both antibiotic production repressors, have been reported so far. ResultsIn this work, we identified a new GBL receptor protein, the conserved and pleiotropic regulator AtrA, which has an activating mode of action. Moreover, we elucidated the precise mechanism by which it controls the production of the antibiotic actinorhodin through the actinorhodin biosynthetic gene cluster activator ActII-orf4. GBL binding to AtrA prevents its binding to the promoter of the actII-orf4 gene, thereby disabling its transcription, while at the same time, GBL binding to ScbR causes coelimycin antibiotic synthesis derepression. ConclusionsThe opposite modes of action of ScbR (repressor) and AtrA (activator) have opposite effects upon GBL binding, activating coelimycin and blocking actinorhodin production at the same time. This phenomenon constitutes an elegant regulatory mechanism that ensures that coelimycin and actinorhodin production are mutually exclusive. These findings also suggest that quorum sensing must be taken into account when designing efficient antibiotic production processes and can be manipulated to ensure both better yield and specificity.

molecular biology↗

bioRxiv2022

Profound immunomodulatory effects of 225Ac-NM600 drive enhanced anti-tumor responsein prostate cancer

An immunosuppressive tumor microenvironment has hampered the efficacy of immunotherapy in prostate cancer. However, radiation-induced immunological effects can partly mediate anti-tumor effects by promoting a pro-inflammatory environment potentially responsive to immunotherapy. Herein, we examined the immunomodulatory properties of a radiopharmaceutical therapy (RPT) with NM600 radiolabeled with either a beta or alpha emitter in two prostate cancer models. 225Ac-NM600, but not 177Lu-NM600, promoted significant anti-tumor effects and improved overall survival. Immunomodulatory effects were dose, radionuclide, and tumor type-dependent. 225Ac-NM600 elicited an array of immunomodulatory effects such as increased CD8/Treg ratio, activation of effector and memory T cells, abrogation of infiltrating suppressor cells (e.g., Tregs and MDSCs), and increased levels of Th1 cytokine and pro-inflammatory chemokines. Importantly, we demonstrate the need to carefully characterize the immune responses elicited by RPT both pre-clinically and clinically to maximize tumor control and avoid potential counterproductive immunosuppressive effects. TeaserTargeted alpha therapy can create a pro-inflammatory tumor micro-environment that partly explains stronger anti-tumor responses in prostate cancer

immunology↗